Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The crude venom of many elapid snakes appeared to contain proteins that activated blood coagulation factor V. The factor V activator present in the venom of Naja naja oxiana was purified to homogeneity by chromatography on a mono-S column. The activator was a single chain protein with an apparent mol. wt of 48,000, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by gel permeation chromatography on Sephacryl S200. Activation of bovine factor V by the purified venom activator was accompanied by proteolytic cleavage of factor V and resulted in the formation of two major polypeptide chains with mol. wts of about 90,000 and 77,000. The final product obtained was compared with thrombin-activated factor V for its ability to function as cofactor in factor Xa-catalysed prothrombin activation in the presence of negatively charged phospholipid vesicles (5 mole% phosphatidylserine/95 mole% phosphatidylcholine). The Km for prothrombin obtained at a saturating amount of venom-activated factor Va was nine-fold higher than with thrombin-activated factor V (0.83 microM vs 0.09 microM, respectively) whereas both factor Va molecules stimulated the Vmax of thrombin formation some 6000-fold. Both forms of factor Va promoted the binding factor Xa to negatively charged phospholipid vesicles. However, the apparent Kd for factor Xa was less favorable in the presence of venom-activated factor V (0.67 x 10(-9) M) than in the presence of thrombin-activated factor V (0.043 x 10(-9) M). Thrombin cleaved a peptide bond in the 77,000 mol. wt polypeptide chain of venom-activated factor V, which resulted in the formation of a normal factor Va light chain. This peptide bond cleavage was, however, not associated with a change of cofactor activity. Venom treatment of thrombin-activated factor V, on the other hand, did remove a small fragment (mol. wt approximately 4000) from the heavy chain of factor Va (94,000), yielding a molecule with reduced cofactor activity. The diminished cofactor activity of venom-activated factor V is, therefore, likely due to the fact that a small peptide fragment, involved in the interaction with prothrombin and factor Xa, is missing from the heavy chain of venom-activated factor V.
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PMID:Activation of bovine factor V by an activator purified from the venom of Naja naja oxiana. 144 Jun 44

Sulfation of human coagulation factor V was investigated by biosynthetically labeling the products of HepG2 cells with [35S]sulfate. There was abundant incorporation of the sulfate label into a product identified as factor V by immunoprecipitation, lability to proteases, affinity for the lectin jacalin, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two or more sites in factor V incorporated sulfate as indicated by labeling of different peptide chains of factor Va. The 150-Kd activation fragment of factor Va incorporated the greatest amounts of sulfate. This fragment of factor Va was bound selectively by jacalin-agarose, reflecting its content of O-linked oligosaccharides. Analysis of an alkaline hydrolysate of sulfate-labeled factor Va by anion-exchange chromatography showed that the sulfate occurred partly in tyrosine sulfate residues and partly in alkaline-labile linkages. Sulfate groups are potentially important structural and functional elements in factor V, and labeling with [35S]sulfate provides a useful approach for examining the biosynthesis and processing of this protein. The hypothesis is advanced that sites of sulfation in factor V and several other plasma proteins contribute to the affinity and specificity of thrombin for these molecules, just as it does for the interaction of thrombin with the potent inhibitor hirudin from leeches.
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PMID:Sulfation of tyrosine residues in coagulation factor V. 216 25

Human coagulation factor V is a protein cofactor that is an essential component of the prothrombinase complex. A full-length factor V cDNA has been subcloned into the mammalian expression vector pDX and used to transfect COS cells. Approximately 95 +/- 4% of the recombinant human factor V (rHFV) synthesized in COS cells is secreted into the culture medium. Forty-eight hours after transfection rHFV antigen levels in the conditioned medium were 70 +/- 15 ng/mL. Factor V activity determined by fibrometer assay increased approximately 5-fold from 0.027 +/- 0.012 to 0.124 +/- 0.044 unit/mL following activation by the factor V activating enzyme from Russell's viper venom (RVV-V). A chromogenic assay specific for factor Va indicated that recombinant factor V had 3.8 +/- 1.3% of the activity of the activated protein. The estimated specific activity of the recombinant factor Va was approximately 1800 +/- 500 units/mg, which is similar to the specific activity of purified plasma factor Va of 1700-2000 units/mg. Immunoprecipitation of [35S]methionine-labeled rHFV revealed a single high molecular mass component (approximately 330 kDa). Treatment of rHFV with thrombin or RVV-V resulted in the formation of proteolytic products that were similar to those seen with plasma factor V. We have also expressed a mutant, rHFV-des-B811-1441, that lacks a large portion of the highly glycosylated connecting region that is present in factor V. Immunoprecipitation of [35S]methionine-labeled rHFV-des-B811-1441 revealed a single-chain polypeptide with Mr approximately 230 kDa. This mutant constitutively expressed 38 +/- 7% of the activity of the RVV-V-activated protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region. 239 12

The effect of human plasmin on human coagulation factor V was studied using isolated proteins. Incubation of factor V with plasmin resulted in a rapid increase in procoagulant activity, followed by a subsequent decline in the ability of factor V to serve as a cofactor in the prothrombinase complex. Identical results were obtained when these reactions were conducted in the presence of dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA), indicating that the changes observed could not have occurred as a consequence of cleavage by alpha-thrombin. Analysis of the products of the reaction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a temporal correlation between the rise and fall in factor V activity and the presence of several transient intermediates. These fragments are distinct from the subunits of alpha-thrombin-activated factor V (factor Va). The activation phase of the reaction was not significantly affected by the presence of phospholipid. In contrast, the rate of degradation of active fragments of factor V and the accompanying loss of activity were markedly enhanced in the presence of phospholipid vesicles. These data suggest that the action of plasmin upon factor V results in the transient formation of proteolytic fragments which express significant procoagulant activity.
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PMID:Activation/inactivation of human factor V by plasmin. 252 Dec 93

The synthesis of coagulation factor V was investigated in isolated rat hepatocytes maintained in long-term primary culture. Two culture conditions were compared. A clotting assay and an immunoprecipitation experiment with rabbit anti rat factor V IgG were used to demonstrate not only the presence of factor V in the cells but also active secretion into the culture medium. Both the inhibition of the clotting reaction in presence of the antibody and absence of thrombin in culture media confirmed the specificity of the clotting assays. Electron microscopic examination located factor V in the endoplasmic reticulum and Golgi apparatus of hepatocytes in common with other liver specific plasma proteins. Examination of liver tissue sections confirmed the production of factor V in hepatocytes but not in hepatic endothelial cells although it did not exclude a transit pathway of factor V through these cells. Addition of Russell viper venom factor V activating enzyme to the culture medium had no effect on the factor V activity. In contrast, treatment of cell extracts did increase the coagulant activity. This suggests that hepatocytes contained principally an unactivated form or procofactor, whereas factor V present into the culture medium was mainly in an activated form. These data provide evidence for synthesis and secretion of an hepatocytic factor V.
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PMID:Biosynthesis of factor V by normal adult rat hepatocytes. 267 84

We have investigated the composition and function of membrane microparticles released from platelets exposed to the C5b-9 proteins of the complement system. Gel-filtered human platelets were incubated with sub-lytic amounts of the purified C5b-9 proteins and the distribution of surface antigens was analyzed using monoclonal antibodies and flow cytometry. C5b-9 assembly caused secretory fusion of the alpha-granule membrane with the plasma membrane and the release of membrane vesicles (approximately 0.1-micron diameter) that contained the plasma membrane glycoproteins (GP) GP Ib and GP IIb-IIIa as well as the alpha-granule membrane protein GMP-140. These microparticles were highly enriched in the C9 neoantigen of the C5b-9 complex. The apparent surface density of C5b-9 on the microparticles was approximately 10(3)-fold higher than on the platelet itself, suggesting that the vesicles were selectively shed from the plasma membrane at the site of C5b-9 insertion. C5b-9 induced the expression of an activation-dependent epitope (recognized by monoclonal antibody, PAC1) in GP IIb-IIIa on the platelet surface but not in GP IIb-IIIa on the microparticles. The surface of the microparticles was also highly enriched in alpha-granule-derived coagulation factor V (or Va), accounting for nearly half of all the membrane-bound factor V detected. The number of potential membrane binding sites for factor Va was probed by adding saturating concentrations of factor Va light chain. Under these conditions, the density of factor Va binding sites on the microparticle surface exceeded that on the C5b-9-treated platelet by three to four orders of magnitude. Moreover, the microparticles provided most of the membrane surface for conversion of prothrombin to thrombin by VaXa. These studies demonstrate that the microparticles shed by C5b-9-treated platelets (and not the platelets themselves) provide the principal binding sites for coagulation factor Va and the principal catalytic surface for the prothrombinase complex. Platelet-derived microparticles formed during complement activation in vivo could provide a membrane surface that facilitates the assembly and dissemination of procoagulant enzyme complexes.
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PMID:Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and express prothrombinase activity. 284 29

The effect of human thrombomodulin isolated from placenta on the procoagulant activity of thrombin was studied and compared to that of rabbit thrombomodulin. The isolated protein was proved to be thrombomodulin because a rabbit antibody against the isolated protein blocked protein C activation by thrombomodulin in solution and also blocked the protein-C-activating cofactor activity of human umbilical vein endothelial cells. The affinity of human thrombomodulin for human thrombin in the presence of fibrinogen is 30 times less than that of rabbit thrombomodulin. This value is based on the measurements of the clotting time of human fibrinogen and thrombin in the presence of increasing amounts of thrombomodulin. Human thrombomodulin was also much less effective compared with rabbit thrombomodulin in inhibiting thrombin-induced human coagulation factor V activation. The ability to inhibit release of [3H]serotonin from washed human platelets was at least 10 times less using human thrombomodulin compared with rabbit thrombomodulin. A partially purified preparation of human lung thrombomodulin was also relatively ineffective in inhibiting thrombin-induced serotonin release from platelets, indicating that the difference between rabbit and human thrombomodulin is one of species rather than of tissue. Thus, while human thrombomodulin is a potent cofactor in protein C activation, it is not an efficient inhibitor of the procoagulant actions of thrombin.
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PMID:Human thrombomodulin is not an efficient inhibitor of the procoagulant activity of thrombin. 298 56

Blood coagulation factor V, the labile factor, is an important cofactor in the activation of prothrombin. Approximately 10 years ago, the first purification procedures for undegraded factor V from bovine and human plasma were reported. This was the starting point for a new area in the research on factor V structure-function relationships. In parallel to this, the structure of the even more labile anti-hemophilic factor (factor VIII) has been elucidated and the two proteins are found to be very similar in structure and in function. In this mini-review, I will focus on work performed in our laboratory, which has led forward to the proposal of a new structural model for factor V. It is based on results obtained with several different techniques, including protein chemistry, DNA technology and high resolution electron microscopy. In plasma, factor V circulates as a single chain, high molecular weight protein. During coagulation a limited number of peptide bonds are cleaved in the factor V molecule by thrombin. This leads to a great increase in biological activity. The active Va species is composed of a noncovalent complex between the N- and C-terminal fragments, whereas the activation fragments correspond to the carbohydrate-rich central portion of the molecule. The activity of factor Va is regulated through the selective degradation of the N-terminal heavy chain fragment by activated protein C. Purified human and bovine factor V was examined by high resolution transmission electron microscopy. Factor V was found to be composed of four major domains, three similar sized globular structures (diameter approx. 80 A) are linked via thin spacers to a larger central domain (diameter approx. 140 A). Activation with thrombin results in a reorganization of the molecule. The thrombin cleavage sites are positioned in the spacers between the different domains and two of the peripheral domains combine to form the active Va species. The new factor V model suggests that a unique and dramatic molecular reorganization occurs during the activation of factor V by thrombin and indicates that the low biological activity of single chain factor V is due to the physical separation of the N- and C-terminal domains by the large central region. Full biological activity can only be expressed after limited proteolysis by thrombin, when the two initially separated domains are free to combine to form the active factor Va molecule.
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PMID:A new model for coagulation factor V suggesting a unique mechanism of activation. 324 83

Purified single-chain human coagulation factor V (Mr approximately 330,000) was visualized by high-resolution transmission electron microscopy. The molecule was found to be composed of four major domains. Three similar sized (approximately 90 X 70 A) globular domains were linked via thin (approximately 30 A) spacers to a somewhat larger (approximately 165 X 138 A) central domain. The center-to-center distances between the larger central domain and each of the peripheral domains were found to be approximately 120 A. Incubation of factor V with thrombin resulted in a separation of the peripheral domains from the central domain. This indicates that the factor V domains now observed correspond to the previously characterized factor V fragments formed by limited proteolysis using thrombin. From these results, a model of the three-dimensional factor V structure, distinct from previous models, is proposed.
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PMID:Ultrastructure of human coagulation factor V. 396 75

Advances in biochemistry, physiology, immunology, and cytochemistry, combined with a variety of new approaches for the evaluation of fine structure, have yielded new insights into the structural physiology and pathology of blood platelets. Subpopulations of platelet granules have been clearly defined; they include the catalase containing organelles referred to as peroxisomes; lysosomes enclosing hydrolytic enzymes; and the alpha-granules in which platelet factor 4, mitogenic factor, beta thromboglobulin, thrombin sensitive protein, fibrinogen, and coagulation factor V are localized. Features of platelet membrane systems have been particularly well-delineated, and recent evidence suggests that membrane complexes serve as the sarcoplasmic reticulum of platelets and the site of prostaglandin synthesis. Improved understanding of platelet biostructure resulting from these observations has made it possible to develop specific relationships between defects in structure and pathological behavior of the cells in vitro and in vivo.
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PMID:Overview article: biostructure of blood platelets. 676


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